Method using short chain peracids for controlling biofuel fermentation process infection and yield loss

ABSTRACT

A process for the use of peracid compositions to eliminate and/or control the growth of undesirable bacteria, including contaminating bacteria, in the fermentation production of alcohol is disclosed. Beneficially, the peracid compositions and methods of use of the same do not interfere or inhibit the growth or replication of yeast and have low or no adverse environmental impact.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.14/457,297 filed Aug. 12, 2014, now U.S. Pat. No. 9,416,375 issued Aug.16, 2016, which is a continuation application of U.S. Ser. No.13/923,465 filed Jun. 21, 2013, now U.S. Pat. No. 8,835,140 issued Sep.16, 2014, which claims priority to provisional application of U.S. Ser.No. 61/662,620 filed Jun. 21, 2012, which are hereby incorporated byreference in their entireties.

BACKGROUND OF THE INVENTION

The biofuel industry employs yeast to covert sugars into ethanol. Aproblem in the industry is that fermentation process equipment and/orthe mash can become contaminated with bacteria that reduce productionyields. As a result, the efficiency of such ethanol fermentation issignificantly limited by other micro-organisms contaminating theprocess, including for example lactic acid and acetic acid bacteria(e.g. yield loss bacterium). Such contaminating bacteria complete forsugar supply with the yeast, resulting in a decrease in ethanolproduction. In addition, the contaminating bacteria can decrease the pHconditions which further inhibit the growth of ethanol-producing yeast.Lactobacillus and Acetobacter are well-known yield loss bacterium in theethanol fermentation industry. As a result, there is a need forpreventing ethanol fermentation yield loss from bacterial infectionintroduced in ethanol fermentation procedures.

Antibiotics are commonly used as a means for controlling unwanted yieldloss bacterium in fermentation plants. However, the byproducts of cornethanol fermentation, including distiller's wet grain solids (i.e.wetcake byproduct) are often used for feed supplies. For example,distiller's grain and dried yeast are frequently used for beef and dairycattle feed. The conventional use of antibiotics, such as virginiamycin,in the ethanol fermentation methods for controlling yield lossundesirably results in the incidental (i.e. sub-therapeutic) dosing ofantibiotics to such animals. Data confirms the survival of antibioticsthrough the distillation process at low levels into the byproducts.There is significant public opposition to such incidental antibioticdosing into animal feed supplies, as well as suggested regulatory (Foodand Drug Administration) consideration for the banning of the use ofantibiotics in the ethanol industry. In addition, the use of antibioticsis costly. Therefore, there is a clear desire to eliminate the use ofsuch antibiotics from the corn ethanol fermentation process.

Antibiotic alternatives have included oxidizing biocides, such asstabilized chlorine dioxide, which may be pumped into a fermenter priorto the fermentable substrate being loaded. During the course offermentation, the organic acids produced by contaminating bacteria arethought to activate the chlorine dioxide in the vicinity of thebacterial cells. Chlorine dioxide has also been used for disinfectingprocess pipes and heat exchangers. Other antibiotic alternatives thathave been employed in limited fashion include inorganic oxidizers, suchas hydrogen peroxide or urea hydrogen peroxide.

A chemical alternative to antibiotics for use in the biofuel industryincludes peracids. Peracids are known for use as sanitizers,disinfectants, deodorizers, and bleaching agents, among other uses.Peracids are particularly well suited for use in both clean-in-placesystems (CIP) and clean-out-of-place systems (COP), as well asindustrial uses including antimicrobial control for washing orprocessing meat surfaces, vegetable fume applications, food and beverageapplications and the like. See U.S. Pat. Nos. 7,498,051, 7,504,123,7,507,429, and 7,569,232, which are herein incorporated by reference intheir entirety. However, such CIP and/or COP methods have not reportedlybeen used in the fermentation industry. Accordingly, it is an objectiveof the claimed invention to develop methods, compositions and systemsfor improved cleaning and sanitation procedures using peracids for usein ethanol fermentation processes.

A further object of the invention is to develop methods, compositionsand systems for improved ethanol yield in ethanol fermentation processesthrough the elimination of unwanted bacterial infiltration.

A further object of the invention is an improved yield loss control forethanol fermentation to replace the convention use of antibiotics in thefield.

BRIEF SUMMARY OF THE INVENTION

An advantage of the invention is a non-antibiotic solution to ethanolfermentation yield management through the use of peracid compositions,namely peroxyoctanoic acids. It is an advantage of the present inventionthat the use of peracid compositions improves ethanol yield withoutcreating any residual animal feed concerns. As a result, the peracidcompositions and methods of employing the same in fermentation processesovercome a significant need in the art for improved sanitization methodsand yield loss management. These and other unexpected benefits achievedby the present invention are disclosed herein.

In an aspect of the invention, a method for reducing and/or eliminatingmicrobial populations in a fermentation system comprising: applying aperacid composition to sanitize a fermentation system, wherein theperacid composition comprises a medium chain peracid and wherein thefermentation system comprises one or more fermentation vessels, pipesand/or components; providing or obtaining a mash sources in saidfermentation system; and reducing and/or eliminating a microbialpopulation of yield loss organisms in said fermentation system.

In a further aspect of the invention, method for reducing yield loss inethanol fermentation processes comprising: applying a peracidcomposition to sanitize a fermentation system, wherein the peracidcomposition comprises a medium chain peracid; fermenting a mash in thepresence of at least a residual portion of said peracid composition andyeast in a vessel of said fermentation system to produce ethanol and asolids content; reducing a microbial population of lactic acid bacteriaand/or acetic acid bacteria; and distilling the fermented mash toseparate at least a portion of the ethanol from said solids content. Ina further aspect, the antimicrobial efficacy of said peracid compositiondoes not interfere with yeast fermentation.

In a still further aspect, the invention discloses a method forreplacing antibiotics in fermentation processes comprising: replacing anantibiotic used in a fermentation process to reduce and/or eliminateLactobacillus and/or Acetobacter species with a peracid composition forapplication to a fermentation system; and introducing the peracidcomposition to a mash source, wherein the peracid composition comprisesfrom about 0.0005 wt-% to about 5 wt-% peroxyoctanoic acid, from about 1wt-% to about 10 wt-% octanoic acid, from about 5 wt-% to about 97 wt-%water, from about 0 wt-% to about 20 wt-% anionic surfactant, from about0 wt-% to about 10 wt-% oxidizing agent; about 0 wt-% to about 35 wt-%inorganic acid, and from about 0 wt-% to about 5 wt-% sequestrant, andwherein the peracid composition does not interfere with yeastfermentation in a fermentation process.

While multiple embodiments are disclosed, still other embodiments of thepresent invention will become apparent to those skilled in the art fromthe following detailed description, which shows and describesillustrative embodiments of the invention. Accordingly, the drawings anddetailed description are to be regarded as illustrative in nature andnot restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a process flow diagram of one or more methods of ethanolproduction including optimal locations for the addition of theperoxyoctanoic acid (or other peracid composition) treatment accordingto the invention employing a peracid composition.

FIG. 2 shows a graph demonstrating the reduction in bacterial count insystems using the peracid compositions compared to control infermentation processes according to the invention.

FIG. 3 shows a graph demonstrating no reduction in yeast in systemsusing the peracid compositions compared to control in fermentationprocesses according to the invention.

FIG. 4 shows a graph demonstrating the reduction in bacterial count insystems using a quaternary compound compositions compared to control infermentation processes according to the invention.

FIG. 5 shows a graph demonstrating no reduction in yeast in systemsusing a quaternary compound compared to control in fermentationprocesses according to the invention.

FIG. 6 shows a graph demonstrating carbon dioxide production in systemstreated with peracid chemistries in the rinse water alone to controlbacterial growth, and photographs of the tested bottles of fermentationliquids.

FIGS. 7A-B and FIGS. 8A-8B show graphs demonstrating the effect ofperacid compositions in the rinse water and impact on total volume ofCO₂ produced or the rate of CO₂ production.

Various embodiments of the present invention will be described in detailwith reference to the drawings, wherein like reference numeralsrepresent like parts throughout the several views. Reference to variousembodiments does not limit the scope of the invention. Figuresrepresented herein are not limitations to the various embodimentsaccording to the invention and are presented for exemplary illustrationof the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention relates to non-antibiotic sanitizing and ethanolyield improvement methods for use in the biofuels industry. The peracidcompositions and methods of employing the same have many advantages overconventional antibiotic methods of controlling yield loss in ethanolfermentation. For example, the use of peracid compositions, namelyperoxyoctanoic acids, providing sanitization benefits for thefermentation system, without causing false positive results in systemcontamination. In addition, the use of peracid compositions improvesethanol yield without creating any residual animal feed concerns.

The embodiments of this invention are not limited to particular methodsand/or peracid compositions for controlling fermentation processes toprevent infection and/or reduce yield losses, which can vary and areunderstood by skilled artisans. It is further to be understood that allterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting in any manner orscope. For example, as used in this specification and the appendedclaims, the singular forms “a,” “an” and “the” can include pluralreferents unless the content clearly indicates otherwise. Further, allunits, prefixes, and symbols may be denoted in its SI accepted form.Numeric ranges recited within the specification are inclusive of thenumbers defining the range and include each integer within the definedrange.

So that the present invention may be more readily understood, certainterms are first defined. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which embodiments ofthe invention pertain. Many methods and materials similar, modified, orequivalent to those described herein can be used in the practice of theembodiments of the present invention without undue experimentation, thepreferred materials and methods are described herein. In describing andclaiming the embodiments of the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

The term “about,” as used herein, refers to variation in the numericalquantity that can occur, for example, through typical measuring andliquid handling procedures used for making concentrates or use solutionsin the real world; through inadvertent error in these procedures;through differences in the manufacture, source, or purity of theingredients used to make the compositions or carry out the methods; andthe like. The term “about” also encompasses amounts that differ due todifferent equilibrium conditions for a composition resulting from aparticular initial mixture. Whether or not modified by the term “about”,the claims include equivalents to the quantities.

The term “actives” or “percent actives” or “percent by weight actives”or “actives concentration” are used interchangeably herein and refers tothe concentration of those ingredients involved in cleaning expressed asa percentage minus inert ingredients such as water or salts.

As used herein, the term “alkyl” or “alkyl groups” refers to saturatedhydrocarbons having one or more carbon atoms, including straight-chainalkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl,octyl, nonyl, decyl, etc.), cyclic alkyl groups (or “cycloalkyl” or“alicyclic” or “carbocyclic” groups) (e.g., cyclopropyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclooctyl, etc.), branched-chain alkyl groups(e.g., isopropyl, tert-butyl, sec-butyl, isobutyl, etc.), andalkyl-substituted alkyl groups (e.g., alkyl-substituted cycloalkylgroups and cycloalkyl-substituted alkyl groups).

Unless otherwise specified, the term “alkyl” includes both“unsubstituted alkyls” and “substituted alkyls.” As used herein, theterm “substituted alkyls” refers to alkyl groups having substituentsreplacing one or more hydrogens on one or more carbons of thehydrocarbon backbone. Such substituents may include, for example,alkenyl, alkynyl, halogeno, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonates, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclic, alkylaryl, or aromatic(including heteroaromatic) groups.

In some embodiments, substituted alkyls can include a heterocyclicgroup. As used herein, the term “heterocyclic group” includes closedring structures analogous to carbocyclic groups in which one or more ofthe carbon atoms in the ring is an element other than carbon, forexample, nitrogen, sulfur or oxygen. Heterocyclic groups may besaturated or unsaturated. Exemplary heterocyclic groups include, but arenot limited to, aziridine, ethylene oxide (epoxides, oxiranes), thiirane(episulfides), dioxirane, azetidine, oxetane, thietane, dioxetane,dithietane, dithiete, azolidine, pyrrolidine, pyrroline, oxolane,dihydrofuran, and furan.

The term “antibiotic,” as used herein, refers to a substance well knownto skilled artisans that controls the growth of bacteria, fungi, orsimilar microorganisms, wherein the substance can be a natural substanceproduced by bacteria or fungi, or a chemically/biochemically synthesizedsubstance (which may be an analog of a natural substance), or achemically modified form of a natural substance.

The term “cellulosic material,” as used herein, refers to materialcontaining cellulose. Cellulose is generally found, for example, in thestems, leaves, hulls, husks, and cobs of plants or leaves, branches, andwood of trees. The cellulosic material can be, but is not limited to,herbaceous material, agricultural residues, forestry residues, municipalsolid wastes, waste paper, and pulp and paper mill residues. It isunderstood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix.

The term “cleaning,” as used herein, means to perform or aid in soilremoval, bleaching, microbial population reduction, or combinationthereof. For the purpose of this patent application, successfulmicrobial reduction is achieved when the microbial populations arereduced by at least about 50%, or by significantly more than is achievedby a wash with water. Larger reductions in microbial population providegreater levels of protection.

As used herein, the term “disinfectant” refers to an agent that killsall vegetative cells including most recognized pathogenicmicroorganisms, using the procedure described in A.O.A.C. Use DilutionMethods, Official Methods of Analysis of the Association of OfficialAnalytical Chemists, paragraph 955.14 and applicable sections, 15thEdition, 1990 (EPA Guideline 91-2). As used herein, the term “high leveldisinfection” or “high level disinfectant” refers to a compound orcomposition that kills substantially all organisms, except high levelsof bacterial spores, and is effected with a chemical germicide clearedfor marketing as a sterilant by the Food and Drug Administration. Asused herein, the term “intermediate-level disinfection” or “intermediatelevel disinfectant” refers to a compound or composition that killsmycobacteria, most viruses, and bacteria with a chemical germicideregistered as a tuberculocide by the Environmental Protection Agency(EPA). As used herein, the term “low-level disinfection” or “low leveldisinfectant” refers to a compound or composition that kills someviruses and bacteria with a chemical germicide registered as a hospitaldisinfectant by the EPA.

The term “Distillers Dried Grains” (DDG), as used herein refersgenerally to coproducts of ethanol production by fermentation which cancomprise dried residual grain solids, which can be animal feed grade.“Distillers Dried Grains with Solubles” (DDGS) refers to coproducts ofethanol production by fermentation which can comprise dried residualgrain solids with solubles content, such as process syrup or othersolubles, and which can be animal feed grade. “Wet Distillers Grains”(WDG) refers to coproducts of ethanol production by fermentation whichcan comprise residual grain solids prior to drying, which can contain atleast a portion of process syrup, and which can be animal feed grade.

As used herein, the term “fermentable sugar” refers to simple sugarssuch as monosaccharides and disaccharides (e.g., glucose (dextrose),fructose, galactose, sucrose, maltose) that can be used by yeast orother microorganisms in conversions to ethanol or other end products.

As used herein, the term “microorganism” refers to any noncellular orunicellular (including colonial) organism. Microorganisms include allprokaryotes. Microorganisms include bacteria (including cyanobacteria),spores, lichens, fungi, protozoa, virinos, viroids, viruses, phages, andsome algae. As used herein, the term “microbe” is synonymous withmicroorganism.

As used herein, the terms “mixed” or “mixture” when used relating to“peroxycarboxylic acid composition” or “peroxycarboxylic acids” refer toa composition or mixture including more than one peroxycarboxylic acid,such as a composition or mixture including peroxyacetic acid (POAA) andperoxyoctanoic acid (POAA).

As used herein, the terms “peracid” or “peroxy acid” refer to an acidhaving the hydrogen of the hydroxyl group replaced by a hydroxy group.Oxidizing peracids are referred to herein as peroxycarboxylic acids.

As used herein the term “peracid forming composition” refers to acomposition that produces a peracid when the components of thecomposition are combined. For example, in some embodiments, a peracidforming composition suitable for use in the present invention includesan organic acid and an oxidizing agent.

For the purpose of this patent application, successful microbialreduction is achieved when the microbial populations are reduced by atleast about 50%, or by significantly more than is achieved by a washwith water. Larger reductions in microbial population provide greaterlevels of protection.

As used herein, the term “sanitizer” refers to an agent that reduces thenumber of bacterial contaminants to safe levels as judged by publichealth requirements. In an embodiment, sanitizers for use in thisinvention will provide at least a 99.999% reduction (5-log orderreduction). These reductions can be evaluated using a procedure set outin Germicidal and Detergent Sanitizing Action of Disinfectants, OfficialMethods of Analysis of the Association of Official Analytical Chemists,paragraph 960.09 and applicable sections, 15th Edition, 1990 (EPAGuideline 91-2). According to this reference a sanitizer should providea 99.999% reduction (5-log order reduction) within 30 seconds at roomtemperature, 25±2° C., against several test organisms.

As used in this invention, the term “sporicide” refers to a physical orchemical agent or process having the ability to cause greater than a 90%reduction (1-log order reduction) in the population of spores ofBacillus cereus or Bacillus subtilis within 10 seconds at 60 C. Incertain embodiments, the sporicidal compositions of the inventionprovide greater than a 99% reduction (2-log order reduction), greaterthan a 99.99% reduction (4-log order reduction), or greater than a99.999% reduction (5-log order reduction) in such population within 10seconds at 60 C.

Differentiation of antimicrobial “-cidal” or “-static” activity, thedefinitions which describe the degree of efficacy, and the officiallaboratory protocols for measuring this efficacy are considerations forunderstanding the relevance of antimicrobial agents and compositions.Antimicrobial compositions can affect two kinds of microbial celldamage. The first is a lethal, irreversible action resulting in completemicrobial cell destruction or incapacitation. The second type of celldamage is reversible, such that if the organism is rendered free of theagent, it can again multiply. The former is termed microbiocidal and thelater, microbistatic. A sanitizer and a disinfectant are, by definition,agents which provide antimicrobial or microbiocidal activity. Incontrast, a preservative is generally described as an inhibitor ormicrobistatic composition

As used herein, the term “substantially free” refers to compositionscompletely lacking the component or having such a small amount of thecomponent that the component does not affect the performance of thecomposition. The component may be present as an impurity or as acontaminant and shall be less than 0.5 wt-%. In another embodiment, theamount of the component is less than 0.1 wt-% and in yet anotherembodiment, the amount of component is less than 0.01 wt-%.

As used herein, the term “sulfoperoxycarboxylic acid,” “sulfonatedperacid,” or “sulfonated peroxycarboxylic acid” refers to theperoxycarboxylic acid form of a sulfonated carboxylic acid. In someembodiments, the sulfonated peracids of the present invention aremid-chain sulfonated peracids. As used herein, the term “mid-chainsulfonated peracid” refers to a peracid compound that includes asulfonate group attached to a carbon that is at least one carbon (e.g.,the three position or further) from the carbon of the percarboxylic acidgroup in the carbon backbone of the percarboxylic acid chain, whereinthe at least one carbon is not in the terminal position. As used herein,the term “terminal position,” refers to the carbon on the carbonbackbone chain of a percarboxylic acid that is furthest from thepercarboxyl group.

As used herein, the term “waters” includes food process or transportwaters. Food process or transport waters include produce transportwaters (e.g., as found in flumes, pipe transports, cutters, slicers,blanchers, retort systems, washers, and the like), belt sprays for foodtransport lines, boot and hand-wash dip-pans, third-sink rinse waters,and the like. Waters also include domestic and recreational waters suchas pools, spas, recreational flumes and water slides, fountains, and thelike.

The term “weight percent,” “wt-%,” “percent by weight,” “% by weight,”and variations thereof, as used herein, refer to the concentration of asubstance as the weight of that substance divided by the total weight ofthe composition and multiplied by 100. It is understood that, as usedhere, “percent,” “%,” and the like are intended to be synonymous with“weight percent,” “wt-%,” etc.

The methods and compositions of the present invention may comprise,consist essentially of, or consist of the components and ingredients ofthe present invention as well as other ingredients described herein. Asused herein, “consisting essentially of” means that the methods andcompositions may include additional steps, components or ingredients,but only if the additional steps, components or ingredients do notmaterially alter the basic and novel characteristics of the claimedmethods, systems, apparatuses, and compositions.

It should also be noted that, as used in this specification and theappended claims, the term “configured” describes a system, apparatus, orother structure that is constructed or configured to perform aparticular task or adopt a particular configuration. The term“configured” can be used interchangeably with other similar phrases suchas arranged and configured, constructed and arranged, adapted andconfigured, adapted, constructed, manufactured and arranged, and thelike.

Compositions

According to an embodiment of the invention a peracid composition isemployed for fermentation procedures, namely in the biofuels industry.In an aspect, the peracid compositions according to the invention mayinclude a peracid (e.g., a peroxycarboxylic acid or asulfoperoxycarboxylic acid) or mixture thereof.

In a further aspect, the peracid composition can also include an organicacid and an oxidizing agent. In a still further aspect, the peracidcomposition can be a peracid forming composition. In various aspects theperacid composition can be formed by an organic acid and an oxidizingagent. In other aspects, peracid forming compositions may be employed togenerate a peracid composition in situ. Additional description ofexemplary in situ methods for peracid forming compositions is providedin U.S. application Ser. Nos. 13/331,304 and 13/331,486 (Ecolab Cases2757USU1 and 2757USU2), which are herein incorporated by reference inits entirety.

The concentration of peracids employed in a peracid compositionaccording to the invention is suitable to replace the antibioticdependent methods of fermentation. In an aspect, the concentration ofperacids is sufficient to sanitize a fermentation system (or portionthereof) or a mash source. In a further aspect, the concentration ofperacids is sufficient to control the problematic yield loss bacteriawithout reducing the yeast required for the ethanol fermentation.

In an aspect, peracid compositions are suitable for use according to themethods of the invention at concentrations employed for surfacedisinfection. For example, concentrations of peracid compositionssuitable for use in non-food contact surface disinfection may beemployed. In a further aspect, peracid compositions can be employed at aconcentration up to about 20,000 ppm. In another aspect, peracidcompositions can be employed at a concentration up to about 10,000 ppm.In a still further aspect, a medium chain peracid composition can beemployed at concentrations from about 2,500 ppm to about 10,000 ppm.Without being according to the invention, all ranges recited areinclusive of the numbers defining the range and include each integerwithin the defined range.

In other aspects according to the invention, more dilute concentrationsof the peracid compositions can be employed. For example, in methodsusing a CIP surface disinfection a surface disinfecting amount of theperacid composition is employed. Thereafter a residual amount of theperacid composition is used as a preservative for a mash source. In anaspect, the peracid compositions are suitable for use according to themethods of the invention at concentrations up to about 2,000. In otheraspects, the residual amounts of the peracid compositions are used atconcentrations from about 1 ppm to about 2,000 ppm, and from about 1 ppmto about 500 ppm. Without being according to the invention, all rangesrecited are inclusive of the numbers defining the range and include eachinteger within the defined range.

The peracid compositions according to the invention do not carry throughthe fermentation and/or distillation process in amounts orconcentrations that cause animal feeding concerns and/or regulatoryconcerns. In addition, the use of peracid compositions do not contributeto public health concerns associated with antibiotic-resistant strainsof bacteria found in the ethanol fermentation process.

Peroxycarboxylic Acids

Peroxycarboxylic (or percarboxylic) acids generally have the formulaR(CO₃H)_(n), where, for example, R is an alkyl, arylalkyl, cycloalkyl,aromatic, or heterocyclic group, and n is one, two, or three, and namedby prefixing the parent acid with peroxy. The R group can be saturatedor unsaturated as well as substituted or unsubstituted. Peroxycarboxylicacids can be made by the direct action of an oxidizing agent on acarboxylic acid, by autoxidation of aldehydes, or from acid chlorides,and hydrides, or carboxylic anhydrides with hydrogen or sodium peroxide.

Peroxycarboxylic acids may include short chain and/or medium chainperoxycarboxylic acids. As used herein, the phrase “medium chainperoxycarboxylic acid” refers to the peroxycarboxylic acid form of amedium chain carboxylic acid. As used herein, the phrase “medium chaincarboxylic acid” refers to a carboxylic acid that: 1) has reduced or islacking odor compared to the bad, pungent, or acrid odor associated withan equal concentration of small chain carboxylic acid, and 2) has acritical micellar concentration greater than 1 mM in aqueous buffers atneutral pH. Medium chain carboxylic acids exclude carboxylic acids thatare infinitely soluble in or miscible with water at 20 C. Medium chaincarboxylic acids include carboxylic acids with boiling points (at 760 mmHg pressure) of 180 to 300° C. In an embodiment, medium chain carboxylicacids include carboxylic acids with boiling points (at 760 mm Hgpressure) of 200 to 300° C. In an embodiment, 20 medium chain carboxylicacids include those with solubility in water of less than 1 g/L at 25°C. Examples of medium chain carboxylic acids include pentanoic acid,hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoicacid, undecanoic acid, and dodecanoic acid.

As used herein, the phrase “short chain peroxycarboxylic acid” refers tothe peroxycarboxylic acid form of a short chain carboxylic acid. As usedherein, the phrase “short chain carboxylic acid” refers to a carboxylicacid that: 1) has characteristic bad, pungent, or acrid odor, and 2) isinfinitely soluble in or miscible with water at 20° C. Examples of shortchain carboxylic acids include formic acid, acetic acid, propionic acid,and butyric acid. In a preferred aspect of the invention, short chaincarboxylic acids are not employed. In particular, acetic acids are notemployed according to a preferred embodiment of the invention. In someembodiments, the compositions and methods of the present invention donot include peroxyacetic acid or acetic acid. Peroxyacetic (orperacetic) acid is a peroxycarboxylic acid having the formula: CH₃COOOH.Generally, peroxyacetic acid is a liquid having an acrid odor at higherconcentrations and is freely soluble in water, alcohol, ether, andsulfuric acid. Peroxyacetic acid can be prepared through any number ofmethods known to those of skill in the art including preparation fromacetaldehyde and oxygen in the presence of cobalt acetate. A solution ofperoxyacetic acid can be obtained by combining acetic acid with hydrogenperoxide.

Peroxycarboxylic acids useful in the compositions and methods of thepresent invention include peroxyformic, peroxyacetic, peroxypropionic,peroxybutanoic, peroxypentanoic, peroxyhexanoic, peroxyheptanoic,peroxyoctanoic, peroxynonanoic, peroxydecanoic, peroxyundecanoic,peroxydodecanoic, or the peroxyacids of their branched chain isomers,peroxylactic, peroxymaleic, peroxyascorbic, peroxyhydroxyacetic,peroxyoxalic, peroxymalonic, peroxysuccinic, peroxyglutaric,peroxyadipic, peroxypimelic and peroxysubric acid and mixtures thereof.

In some embodiments, the compositions of the invention utilize acombination of several different peroxycarboxylic acids. For example, insome embodiments, the composition includes one or more C1 to C4peroxycarboxylic acids and one or more C5 to C11 peroxycarboxylic acids.Especially preferred is an embodiment in which the C1 to C4peroxycarboxylic acid is peroxyacetic acid and the C5 to C11 acid isperoxyoctanoic acid.

In some embodiments, the compositions and methods of the presentinvention include peroxyoctanoic acid. Peroxyoctanoic (or peroctanoic)acid is a peroxycarboxylic acid having the formula, for example, ofn-peroxyoctanoic acid: CH₃(CH₂)₆COOOH. Peroxyoctanoic acid can be anacid with a straight chain alkyl moiety, an acid with a branched alkylmoiety, or a mixture thereof. Peroxyoctanoic acid is surface active andcan assist in wetting hydrophobic surfaces, such as those of microbes.Peroxyoctanoic acid can be prepared through any number of methods knownto those of skill in the art. A solution of peroxyoctanoic acid can beobtained by combining octanoic acid and hydrogen peroxide. In an aspectof the invention a commercially-available peroxyoctanoic acid containingproduct is available under the commercial name Octave (Ecolab, Inc.).Additional description of particularly suitable peroxyoctanoic acids isdisclosed in U.S. Pat. Nos. 7,498,051, 7,504,123, 7,507,429 and7,569,232, which are herein incorporated by reference.

In a preferred aspect of the invention, the peracid does not include aperacetic acid and/or acetic acid (organic acid source). There is anadditional unexpected benefit of employing a peroxyoctanoic acidaccording to an embodiment of the invention. In particular, use of theperoxyoctanoic acid avoids introduction of acetic acid, which is commonto many commercial peracid sanitizing compositions. Acetic acid has beendemonstrated to detrimentally result in false positive tests on qualitycontrol applications; the organic acid appears on plant quality controltests (e.g. Gas Chromatography) as a false indicator of infection withacid-forming bacteria. As a result the peracid compositions and methodsof employing the same according to the invention that employs aperoxyoctanoic acid composition due to result in false positiveindicators of acid-forming bacteria infecting a fermentation system.

In some embodiments, the compositions of the present invention includeabout −0.0005 wt-% to about 20 wt-%, about 0.3 wt-% to about 10 wt-%,about 0.5 wt-% to about 5.0 wt-%, about 1 wt-% to about 3 wt-%, or about1 wt-% to about 2 wt-% of one or more peroxycarboxylic acids. It is tobe understood that all values and ranges between these values and rangesare encompassed by the present invention.

Sulfoperoxycarboxylic Acids

Sulfoperoxycarboxylic (or sulfopercarboxylic) acids generally have theformula

wherein R₁ is hydrogen, or a substituted or unsubstituted alkyl group;R₂ is a substituted or unsubstituted alkyl group; X is hydrogen, acationic group, or an ester forming moiety; or salts or esters thereof.In some embodiments, R₁ is a substituted or unsubstituted C_(m) alkylgroup; X is hydrogen a cationic group, or an ester forming moiety; R₂ isa substituted or unsubstituted C_(n) alkyl group; m=1 to 10; n=1 to 10;and m+n is less than 18, or salts, esters or mixtures thereof. In someembodiments, R₁ is hydrogen. In other embodiments, R₁ is a substitutedor unsubstituted alkyl group. In some embodiments, R₁ is a substitutedor unsubstituted alkyl group that does not include a cyclic alkyl group.

In some embodiments, R₁ is a substituted alkyl group. In someembodiments, R₁ is an unsubstituted C₁-C₉ alkyl group. In someembodiments, R₁ is an unsubstituted C₇ or C₈ alkyl. In otherembodiments, R₁ is a substituted C₈-C₁₀ alkyl group.

In some embodiments, R₁ is a substituted C8-C10 alkyl group issubstituted with at least 1, or at least 2 hydroxyl groups. In still yetother embodiments, R₁ is a substituted C1-C9 alkyl group. In someembodiments, R₁ is a substituted C1-C9 substituted alkyl group issubstituted with at least 1 SO₃H group.

In other embodiments, R₁ is a C9-C10 substituted alkyl group. In someembodiments, R₁ is a substituted C9-C10 alkyl group wherein at least twoof the carbons on the carbon backbone form a heterocyclic group. In someembodiments, the heterocyclic group is an epoxide group.

In some embodiments, R₂ is a substituted C1 to C10 alkyl group. In someembodiments, R₂ is a substituted C8-C10 alkyl. In some embodiments, R₂is an unsubstituted C6-C9 alkyl. In other embodiments, R₂ is a C8 to C10alkyl group substituted with at least one hydroxyl group. In someembodiments, R₂ is a C10 alkyl group substituted with at least twohydroxyl groups. In other embodiments, R₂ is a C8 alkyl groupsubstituted with at least one SO₃H group. In some embodiments, R₂ is asubstituted C9 group, wherein at least two of the carbons on the carbonbackbone form a heterocyclic group. In some embodiments, theheterocyclic group is an epoxide group. In some embodiments, R₁ is aC8-C9 substituted or unsubstituted alkyl, and R₂ is a C7-C8 substitutedor unsubstituted alkyl.

Additional description of particularly suitable sulfoperoxycarboxylicacids is disclosed in U.S. Pat. No. 8,344,026 and U.S. patentapplication Ser. Nos. 12/568,493 and 13/290,355 which are hereinincorporated by reference in their entirety.

Without wishing to be bound by any particular theory, it is thought thatmid-chain sulfonated peracids, e.g., mid-chain sulfonated peracids witha C10-C18 carbon backbone have a substantially greater solubilitycompared to terminally sulfonated peracids of a similar chain length,even at an acidic pH. For example, at a pH of 4, the terminallysulfonated peracid, 11-sulfoundecane peroxoic acid has a relatively lowsolubility of about 1.3%. At the same pH, the mid chain sulfonatedperacid, persulfonated oleic acid has a solubility of greater than about50%. This is unexpected as an increase in peracid chain length isthought to lead to a decrease in solubility. The issue of low solubilitywhen using long chain peracids has been addressed by increasing the pHto above 7. However, at increased pH antimicrobial efficacy issubstantially reduced. Further, bleaching efficacy decreasesproportionally with every pH unit increase over about 7. Thus,solubility at an acidic pH (lower than about 7) is beneficial to themid-chain sulfonated peracids of the present invention.

In some embodiments, the compositions of the invention utilize acombination of several different sulfoperoxycarboxylic acids. Forexample, in some embodiments, the composition includes one or more C1 toC4 sulfoperoxycarboxylic acids and one or more C5 to C11sulfoperoxycarboxylic acids.

The sulfoperoxyacids disclosed according to the invention can be formedusing a variety of reaction mechanisms. For example, in someembodiments, the peracids are formed by the direct acid catalyzedequilibrium action of hydrogen peroxide with the starting materials.

In some embodiments, the compositions of the present invention includeabout −0.0005 wt-% to about 20 wt-%, about 0.3 wt-% to about 10 wt-%,about 0.5 wt-% to about 5.0 wt-%, about 1 wt-% to about 3 wt-%, or about1 wt-% to about 2 wt-% of one or more sulfoperoxycarboxylic acids. It isto be understood that all values and ranges between these values andranges are encompassed by the present invention.

Organic Acids

The peracid compositions may also include at least one organic acid. Anyorganic acid capable of forming a peracid can be used in thecompositions and methods of the present invention. Suitable organicacids for use with the present invention include, but are not limitedto, carboxylic acids.

In some embodiments, the compositions of the present invention includeat least one carboxylic acid. In some embodiments, the compositions ofthe present invention include at least two, at least three, or at leastfour or more carboxylic acids.

In some embodiments, the carboxylic acid for use with the compositionsof the present invention is a C1 to C22 carboxylic acid. In someembodiments, the carboxylic acid for use with the compositions of thepresent invention is a C5 to C11 carboxylic acid. In some embodiments,the carboxylic acid for use with the compositions of the presentinvention is a C1 to C4 carboxylic acid. Examples of suitable carboxylicacids include, but are not limited to, formic, acetic, propionic,butanoic, pentanoic, hexanoic, heptanoic, octanoic, nonanoic, decanoic,undecanoic, dodecanoic, as well as their branched isomers, lactic,maleic, ascorbic, citric, hydroxyacetic, neopentanoic, neoheptanoic,neodecanoic, oxalic, malonic, succinic, glutaric, adipic, pimelic subricacid, and mixtures thereof.

In some embodiments, the compositions of the present invention includeabout 10 wt-% to about 95 wt-%, about 25 wt-% to about 80 wt-%, or about50 wt-% to about 75 wt-% of a carboxylic acid. In some embodiments, thecompositions of the present invention include acetic acid. In otherembodiments, the compositions of the present invention include octanoicacid. In other embodiments, the compositions of the present inventioninclude a combination of octanoic acid and acetic acid.

Oxidizing Agent

The peracid compositions may also include an oxidizing agent. Theoxidizing agent can be effective to convert an acid into a peracid. Theoxidizing agent may include a peroxide source. Oxidizing agents suitablefor use with the compositions include the following types of compoundsor sources of these compounds, or alkali metal salts including thesetypes of compounds, or forming an adduct therewith: hydrogen peroxide,urea-hydrogen peroxide complexes or hydrogen peroxide donors of: group 1(IA) oxidizing agents, for example lithium peroxide, sodium peroxide;group 2 (IIA) oxidizing agents, for example magnesium peroxide, calciumperoxide, strontium peroxide, barium peroxide; group 12 (IIB) oxidizingagents, for example zinc peroxide; group 13 (IIIA) oxidizing agents, forexample boron compounds, such as perborates, for example sodiumperborate hexahydrate of the formula Na₂[B₂(O₂)₂(OH)₄].6H₂O (also calledsodium perborate tetrahydrate); sodium peroxyborate tetrahydrate of theformula Na₂B₂(O₂)₂[(OH)₄].4H₂O (also called sodium perboratetrihydrate); sodium peroxyborate of the formula Na₂[B₂(O₂)₂(OH)₄] (alsocalled sodium perborate monohydrate); group 14 (WA) oxidizing agents,for example persilicates and peroxycarbonates, which are also calledpercarbonates, such as persilicates or peroxycarbonates of alkalimetals; group 15 (VA) oxidizing agents, for example peroxynitrous acidand its salts; peroxyphosphoric acids and their salts, for example,perphosphates; group 16 (VIA) oxidizing agents, for exampleperoxysulfuric acids and their salts, such as peroxymonosulfuric andperoxydisulfuric acids, and their salts, such as persulfates, forexample, sodium persulfate; and group VIIa oxidizing agents such assodium periodate, potassium perchlorate. Other active inorganic oxygencompounds can include transition metal peroxides; and other suchperoxygen compounds, and mixtures thereof.

In some embodiments, the compositions of the present invention employone or more of the inorganic oxidizing agents listed above. Suitableinorganic oxidizing agents include ozone, hydrogen peroxide, hydrogenperoxide adduct, group IIIA oxidizing agent, or hydrogen peroxide donorsof group VIA oxidizing agent, group VA oxidizing agent, group VIIAoxidizing agent, or mixtures thereof. Suitable examples of suchinorganic oxidizing agents include percarbonate, perborate, persulfate,perphosphate, persilicate, or mixtures thereof.

In some embodiments, the oxidizing agent includes hydrogen peroxide, ora source or donor of hydrogen peroxide. Hydrogen peroxide can beprovided as a mixture of hydrogen peroxide and water, e.g., as liquidhydrogen peroxide in an aqueous solution. Hydrogen peroxide iscommercially available at concentrations of 35%, 70%, and 90% in water.

The compositions may contain an effective amount of an oxidizing agent.In some embodiments, the compositions include about 0.001 wt-% to about60 wt-% of the oxidizing agent, or about 1 wt-% to about 25 wt-% of theoxidizing agent. In some embodiments, the compositions include about 30wt-% to about 50 wt-% of the oxidizing agent. It is to be understoodthat all ranges and values between these ranges and values areencompassed by the present invention.

Solubilizer

The present peracid compositions can include a solubilizer. The presentinvention relates to solubilizers for various peroxycarboxylic acids,including preferably peroxyoctanoic acid. In an embodiment, thesolubilizer can increase or maintain the solubility in the composition.The present compositions and methods can include any of a variety ofsuitable solubilizers. For example, the solubilizer can include asolvent, a surfactant, or a mixture thereof as disclosed herein. Furtherdescription of solubilizers particularly well suited for use withperoxyoctanoic acid compositions is found in U.S. Pat. Nos. 7,498,051and 7,569,232, which are herein incorporated by reference in theirentirety.

Solvent

In some embodiments, the peracid compositions of the present inventionare liquids. Therefore, in some embodiments, the compositions of theinvention further include a solvent or solubilizer. In some embodiments,the solvent is water. The water may be provided by the use of aqueousreagents, viz. oxidizing agent. In other embodiments, an additionalamount of water is added to the peracid compositions.

In some embodiments, the liquid composition according to the presentinvention is a composition including more than 10 wt-% water but lessthan 90 wt-%. The amount of water included in the liquid composition canbe for example, less than about 80 wt-%, less than about 70 wt-%, andless than about 60 wt-% by weight of the liquid composition. In someembodiments, the composition can contain water between about 5 wt-% andabout 50 wt-%, about 10 wt-% and about 40 wt-%, or about 30 wt-%. It isto be understood that all values and ranges between these values andranges are encompassed by the methods of the present invention.

Alternatively, the compositions may be free of or substantially free ofany added water. A non-aqueous solvent may also be used in thecompositions. For example, in some embodiments, an alcohol is includedas a solvent in the compositions. In some embodiments, a liquidcomposition of the invention is substantially non-aqueous (or anhydrous)in character. The term “substantially non-aqueous” as used herein meansthat while very small amounts of water may be incorporated into suchpreferred compositions, the amount of water in the non-aqueous liquiddetergent compositions of the invention are less than about 30 wt-% ofthe composition. In some embodiments, the water content of thenon-aqueous compositions will include less than about 10 wt-% by weight.

The compositions may include an effective amount of solvent. In someembodiments, the compositions may include about 10 wt-% to about 99 wt-%of a solvent, or about 20 wt-% to about 80 wt-% of a solvent. In otherembodiments, the compositions may include more than about 30 wt-%, morethan about 50 wt-%, more than about 60 wt-% or more than 70% of asolvent. It is to be understood that all values and ranges between thesevalues and ranges are encompassed by the present invention.

Additional Functional Ingredients

The peracid compositions may also include additional functionalingredients. Additional functional ingredients suitable for use in thepresent compositions include, but are not limited to, stabilizingagents, surfactants, acidulants, hydrotropes, dispersants, antimicrobialagents, optical tracers, solidification agent, aesthetic enhancing agent(i.e., colorant (e.g., pigment), odorant, or perfume), wetting agents,defoaming agents, thickening or gelling agents, among any number ofconstituents which can be added to the composition. Such adjuvants canbe preformulated with the peracid compositions or added to thecompositions after formation, but prior to use. The compositions canalso contain any number of other constituents as necessitated by theapplication, which are known and which can facilitate the activity ofthe present compositions.

Stabilizing Agents

Stabilizing agents are commonly added to equilibrium peracidcompositions to stabilize the peracid and hydrogen peroxide and preventthe decomposition of these constituents. Examples of stabilizing agentsmay include for example, surfactants, couplers, hydrotropes, acidcatalysts and the like that are conventionally used in equilibriumperacid compositions to stabilize and improve shelf life of thecomposition. Further examples of stabilizing agents include, forexample, chelating agents or sequestrants. Such sequestrants include,but are not limited to, organic chelating compounds that sequester metalions in solution, particularly transition metal ions. Such sequestrantsinclude organic amino- or hydroxy-polyphosphonic acid complexing agents(either in acid or soluble salt forms), carboxylic acids (e.g.,polymeric polycarboxylate), hydroxycarboxylic acids, aminocarboxylicacids, or heterocyclic carboxylic acids, e.g., pyridine-2,6-dicarboxylicacid (dipicolinic acid). Dipicolinic acid, 1-hydroxyethylidene-1,1-diphosphonic acid (CH₃C(PO₃H₂)₂OH) (HEDP) are furtherexample of stabilizing agents.

Additional examples of stabilizing agents commonly used in equilibriumchemistry to stabilize the peracid and hydrogen peroxide and/or preventthe premature oxidation of the composition include phosphonic acid orphosphonate salt. Phosphonic acids and phosphonate salts include HEDP;ethylenediamine tetrakis methylenephosphonic acid (EDTMP);diethylenetriamine pentakis methylenephosphonic acid (DTPMP);cyclohexane-1,2-tetramethylene phosphonic acid; amino[tri(methylenephosphonic acid)]; (ethylene diamine[tetra methylene-phosphonic acid)];2-phosphene butane-1,2,4-tricarboxylic acid; or salts thereof, such asthe alkali metal salts, ammonium salts, or alkyloyl amine salts, such asmono, di, or tetra-ethanolamine salts; picolinic, dipicolinic acid ormixtures thereof. In some embodiments, organic phosphonates, e.g., HEDPare well known as used stabilizing agents.

Surfactants

In some embodiments, the peracid compositions of the present inventionmay include a surfactant. Surfactants may be included as a solubilizerfor the peracid compositions (e.g. microemulsion forming surfactant).Surfactants suitable for use with the compositions of the presentinvention include, but are not limited to, anionic surfactants, nonionicsurfactants, cationic surfactants, amphoteric surfactants, zwitterionicsurfactants, mixtures thereof, or the like.

The solubilizer can include a microemulsion forming surfactant. Suitablemicroemulsion forming surfactants include anionic surfactants, cationicsurfactants, amphoteric surfactants, zwitterionic surfactants, mixturesthereof, or the like. Suitable microemulsion forming surfactants includeanionic surfactants, such as sulfate surfactant, sulfonate surfactant,phosphate surfactant (phosphate ester surfactant), and carboxylatesurfactant, mixtures thereof, or the like.

Preferred Compositions

In an aspect of the invention preferred peracid compositions for useaccording to the invention comprise, consist of and/or consistessentially of: a medium chain peroxycarboxylic acid. The medium chainperoxycarboxylic acid may include peroxyoctanoic acid; octanoic acid;and water. The medium chain peroxycarboxylic acid may also include astabilizer, such as polyalkylene oxide, mono alkyl ether of polyalkyleneoxide, dialkyl ether of polyalkylene oxide, nonionic surfactant, anionicsurfactant, or mixture thereof. In a preferred aspect, the compositionsinclude at least about 2 parts by 35 weight of peroxyoctanoic acid foreach 7 parts by weight of octanoic acid.

In additional aspects, the preferred peracid compositions may comprise,consist of and/or consist essentially of: a medium chainperoxycarboxylic acid composition. The medium chain peroxycarboxylicacid composition may include a peroxycarboxylic acid that includes about0.0005 wt-% to about 5 wt-% peroxyoctanoic acid; about 0.001 wt-% toabout 10 wt-% octanoic acid; and about 5 wt-% to about 99.99 wt-% water.

In additional aspects, the preferred peracid compositions may comprise,consist of and/or consist essentially of: a medium chainperoxycarboxylic acid composition. The medium chain peroxycarboxylicacid composition may include about 0.5 wt-% to about 5 wt-%peroxyoctanoic acid; about 1 wt-% to about 10 wt-% octanoic acid; about5 wt-% to about 97 wt-% water; about 1 wt-% to about 20 wt-% anionicsurfactant; about 5 wt-% to about 10 wt-% hydrogen peroxide and/or apersulfate; about 15 wt-% to about 35 wt-% inorganic acid (e.g. sulfuricacid or methanesulfonic acid); and about 1 wt-% to about 5 wt-%sequestrant; the composition comprising a microemulsion.

In additional aspects, the preferred peracid compositions may comprise,consist of and/or consist essentially of: a medium chainperoxycarboxylic acid composition. The medium chain peroxycarboxylicacid composition may include about 0.5 wt-% to about 5 wt-%peroxyoctanoic acid; about 1 wt-% to about 10 wt-% octanoic acid; about5 wt-% to about 97 wt-% water; about 1 wt-% to about 20 wt-% anionicsurfactant; about 5 wt-% to about 10 wt-% oxidizing agent; about 15 wt-%to about 35 wt-% inorganic acid; and about 1 wt-% to about 5 wt-%sequestrant; the composition comprising a microemulsion.

In additional aspects, the preferred peracid compositions may comprise,consist of and/or consist essentially of: a medium chainperoxycarboxylic acid composition. The medium chain peroxycarboxylicacid composition may include about 0.0005 wt-% to about 5 wt-%peroxyoctanoic acid; about 0.001 wt-% to about 10 wt-% octanoic acid;about 40 wt-% to about 99.99 wt-% water; about 0.001 wt-% to about 60wt-% polyalkylene oxide, monoalkyl ether of polyalkylene oxide, dialkylether of polyalkylene oxide, anionic surfactant, nonionic surfactant, ormixture thereof, or mixture thereof; about 0.002 wt-% to about 10 wt-%oxidizing agent; about 0.001 wt-% to about 30 wt-% inorganic acid; andabout 0.001 wt-% to about 5 wt-% sequestrant.

The preferred compositions for use in the methods of the inventionprovide the various benefits not previously achieved by others employingperacid compositions. See U.S. patent application Ser. No. 13/048,972(Buckman Laboratories International, Inc.), which is herein incorporatedby reference. The prior art employing certain nonoxidizing biocides,including peracetic acid, has not employed the superior efficacy of themedium chain peroxycarboxylic acids, namely peroxyoctanoic acidcompositions. In addition, the methods of application and use disclosedin the invention represent additional improvements over the art.

Systems for Ethanol Fermentation

Systems and methods for ethanol fermentation are well known in the art.In general, the processes for converting a complex carbohydrate orstarch to fermentable sugar includes the following steps: a grain orcereal containing granular starch (e.g. cellulosic material such ascorn) is obtained; a milling step is employed (e.g. wet milling or drymilling); mixing milled starch-containing material with an aqueoussolution to produce a slurry; liquefaction processing (if required);addition of a suitable enzyme (if required); saccharification;purification (if required); and finally metabolization/fermentation by afermenting microorganism (e.g. yeast) to create ethanol. As one of skillin the art appreciates, the saccharification and fermentation steps maybe carried out sequentially or simultaneously. The final step is thedistillation of the fermented mass to separate ethanol product from theremaining byproduct (e.g. stillage), which can be processed to formdistillers dried grains.

The methods of the invention that are disclosed herein are suitable foruse in various applications of fermentation methods. This includesvarying the sources of cellulosic materials or feedstock materials. Forexample, without being limited to a particular theory of the invention,the present invention using peracid compositions for yield loss bacteriacontrol can be used without limitation with respect to the feedstocksource for the methods of ethanol fermentation.

Examples of suitable feedstock sources, include for example,agricultural crops, such as grains (e.g., corn, wheat, grain sorghum,barley, rice, sugar cane, and the like); agricultural waste associatedwith crops; lignocellulosic biomasses (e.g. wood chips, corn stover,corn cobs, straw, grain hulls, recycled papers and the like); fruitsand/or fruit juices; refined sugars; combinations of the same; and otherfeedstock sources appreciated by those skilled in the art.

Further description of the methods and system set-up customarilyemployed in ethanol fermentation are shown in FIG. 1, which is anon-limiting embodiment of systems according to the invention. Asdemonstrated in FIG. 1, the peracid compositions, such as aperoxyoctanoic acid composition, may be added into an ethanol processwithin a CIP process, directed additive, or both. As is described inmore detail herein, the use of peracid compositions according to theinvention are particularly suitable for cleaning the apparatus/systemsemployed for the ethanol process (e g tanks, cookers, fermentationvessels and the like). In addition, according to various embodiments ofthe invention the peracid compositions may be employed upstream from thefermentation vessels of the system.

Methods

The methods employing the peracid compositions according to theinvention are suitable for various applications in fermentationprocesses and systems, namely in the biofuels industry. Fermentationprocesses may include, for example, ethanol plants which may use avariety of substrates such as corn and cane sugar. For example, it iscontemplated that the peracid compositions are suitable for systemsanitation (e.g. tank/vessel sanitation) as well as a process aid toimprove fermentation yield without the introduction of antibiotics.

According to an embodiment the invention, methods for reducing and/oreliminating microbial populations in a fermentation system are provided.In some aspects, the methods of treating microbial populations areeffective for killing one or more of the pathogenic bacteria associatedwith ethanol fermentation methods. Such bacteria include a wide varietyof microorganisms, such as aerobic and anaerobic bacteria, includingGram positive and Gram negative bacteria, yeast, molds, bacterialspores, viruses, etc. In particular, the methods of the invention areparticularly suitable for use against lactic acid bacteria. “Lactic acidbacteria,” as used herein, refers to a class of bacteria including, forexample, Lactobacillus, Pediococcus, Leuconostoc and Weissella species.Acetic acid bacteria, e.g., Acetobacter species, can also cause problemsby producing acetic acid or other organic acids which foul the processand reduce the yields of ethanol.

In a preferred aspect of the invention, the peracid compositions andmethods of employing the same for reducing and/or eliminating microbialpopulations in a fermentation system do not interference with yeastperformance.

According to an embodiment the invention, methods for reducing yieldloss in ethanol fermentation are provided. In an aspect of the inventionthe use of peracid compositions according to the invention are employedto reduce and/or eliminate bacterial infiltration or infection of afermentation system. The peracid compositions are employed for use as asurface sanitizer.

According to a further embodiment, methods for replacing antibiotics inethanol fermentation processes are provided. In an aspect, anon-antibiotic dependent method for controlling yield loss in afermentation process is provided. In an embodiment, the use of a peracidcomposition in the ethanol fermentation process replaces theconventional use of antibiotics. Still further, the use of the peracidcompositions according to these methods not only provide a high degreeof antimicrobial efficacy, the resultant fermentation byproducts arefurther safely ingested by animals as a feed supply while imposing nounacceptable environmental incompatibility.

The various methods of the invention include may comprise, consist ofand/or consist essentially of one or more of the following steps:sanitizing a fermentation vessel; sanitizing one or more fermentationvessels, pipes and/or components (including downstream equipmentemployed for fermentation); sanitizing a fermentation source (e.g. mash)or other component with a peracid composition; and the like.

In an aspect of the invention, a peracid composition is introduced intoa vessel or system/apparatus employed for ethanol fermentation tosanitize the surface against unwanted bacterial agents, namely yieldloss agents. The introduction of the peracid composition is employed forhard surface cleaning and sanitizing, which may include clean-in-placesystems (CIP) and/or clean-out-of-place systems (COP). For ethanolfermentation processes, COP systems may include for example readilyaccessible systems including wash tanks/vessels, fermentation vessels,other tanks/vessels, removable system parts, and the like. For ethanolfermentation processes, CIP systems may include the internal componentsof tanks, lines, pumps and other process equipment used for processingtypically liquid product streams. Beneficially, the treatment of thevarious CIP and/or COP portions of the system are uniquely suited to thefermentation systems which rely heavily on internal recycling offermentation components. Such internal recycling is well suited to themethods of the invention employing peracid compositions as thesecompositions that have sufficient longevity and compatibility with thefermentation source/materials.

In a preferred aspect, the peracid composition is introduced (e.g.injected) into a fermentation vessel (i.e. fermentor). In a furtheraspect, the peracid composition is introduced upstream from afermentation tank, (e.g. in the mash cooler where bacterial infectionsoften reside). Such introduction may further be in combination withtraditional cleaning and sanitation practices that are routinelyperformed on the fermentation tank. The introduction of a peracidcomposition upstream from a fermentation tank may be combined with outCIP and/or CIP systems described herein.

In a further aspect, the peracid composition (or a portion thereof)remains in the vessel or fermentation system instead of being drainedtherefrom. The amount of peracid composition remaining in the vessel mayvary according to the desired level of sanitization and dependent uponthe stability of the peracid composition (including amount of peracidcomposition remaining in byproducts of the ethanol fermentation, e.g.distiller's grain). The residual or remaining peracid composition can bebeneficially included with a mash source and act as a preservative forthe fermentation process. The use of a residual amount of a peracidcomposition in a mash source of the fermentation process providesadditional benefits as the mash source is a common infection point forthe fermentation process.

Beneficially and unexpectedly, there methods of the invention do notrequire a rinse step to completely remove the peracid compositions. Manysanitizing methods employ a rinse step, such as rinsing an apparatuswith other materials such as potable water.

It is to be understood that the methods may employ an aqueous ornon-aqueous peracid composition. In addition, either a concentrate oruse concentration of the peracid compositions can be applied to orbrought into contact with an object by any conventional method orapparatus for applying an antimicrobial or cleaning compound to anobject, such as disclosed for example in applications of use describedin U.S. Pat. No. 7,507,429, which is herein incorporated by reference inits entirety. For example, the object can be wiped with, sprayed with,poured on, foamed on, and/or immersed in the compositions, or a usesolution made from the compositions. The compositions can be sprayed,foamed, or wiped onto a surface; the compositions can be caused to flowover the surface, or the surface can be dipped into the compositions.These and other methods of contacting an object or a surface with theperacid composition are within the scope of the invention. Contactingcan be manual or by machine.

The onsite production of the peracid composition is also included withinthe scope of the invention. Exemplary methods and/or apparatus forproducing certain peracid compositions are disclosed for example in U.S.patent application Ser. Nos. 13/330,915, 13/330,981, 13/331,104,13/331,304, 13/331,385 and 13/331,486 (Ecolab 2757USU1, 2757USU2,2839USU1, 2943US01, 2969US01 and 2997US01), which are incorporatedherein by reference in their entirety.

The methods may include the introduction of the peracid compositions ata temperature in the range of about 4° C. to 60° C. After introductionof the peracid composition, the peracid (e.g. solution) is held in thevessel and/or circulated throughout the system for a time sufficient forsanitization (e.g., to kill undesirable microorganisms). The contacttime can vary based upon the concentration of the peracid compositions,method of applying the peracid compositions, temperature conditions,amount of soil, microorganisms or contamination on the surface orapparatus to be treated, or the like. In some aspects of the invention,the exposure time can be at least about 5 seconds, at least about 15seconds, or more. In other embodiments, the exposure time is at least afew minutes. After the surfaces have been sanitized by means of theperacid compositions, the solution may be removed (e.g. drained from thesystem) or retained (in whole or in part) in the system for additionalsanitizing benefit.

In some embodiments, the methods of the invention may further employpressure and/or mechanical action with the application of the peracidcomposition. As one of skill in the art will appreciate, mechanicalaction may include for example, agitation, rubbing, brushing, etc.Agitation can be by physical scrubbing of the surface (e.g. vessel),through the action of the spray solution under pressure, throughsonication, or by other methods. Agitation increases the efficacy of thespray solution in killing micro-organisms, perhaps due to betterexposure of the solution into the crevasses or small colonies containingthe micro-organisms. The spray solution, before application, can also beheated to a temperature of about 15 to 20° C., for example, about 20 to60° C. to increase efficacy.

As one of skill in the art will ascertain as being within the scope ofthe invention, the amount of peracid composition provided to afermentation system will vary based upon a number of factors. Forexample, the size, structural orientation, materials employed therein,contamination level of the system, and the like will affect the amount(and/or concentration) of peracid composition applied thereto. In someaspects, hundreds of gallons of peracid composition (e.g. solution) maybe provided to a fermentation system. In other aspects, thousands ofgallons of peracid composition (e.g. solution) may be provided to afermentation system, including for CIP cleaning methods.

Further demonstration of the methods according to the invention is shownin FIG. 1 (referenced above).

All publications and patent applications in this specification areindicative of the level of ordinary skill in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated as incorporated by reference.

EXAMPLES

Embodiments of the present invention are further defined in thefollowing non-limiting Examples. It should be understood that theseExamples, while indicating certain embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theembodiments of the invention to adapt it to various usages andconditions. Thus, various modifications of the embodiments of theinvention, in addition to those shown and described herein, will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

Example 1

Initial laboratory testing to confirm the performance of peracidcompositions to suppress Lactobacillus species and Acetobacter speciesin the fermentation process was conducted. The biocide fermentationtests were conducted using Vortexx ESF (peracetic acid+octanoic acid)and Whisper V (quaternary ammonium compound) each commercially-availablefrom Ecolab, Inc. The methods of the testing included:

Obtaining fermentation flasks containing 100 ml of fermentation mixture(10% of yeast+Lactobacillus brevis infection (ATCC Deposit No.14869)+brown sugar juice 19° Brix).

-   -   At Time zero (T=0)→10% of yeast+Lactobacillus infection (ATCC        Deposit No. 14869)+brown sugar juice 19° Brix.    -   One hour after biocide addition in the flask (T=1)→10% of        yeast+Lactobacillus infection (ATCC Deposit No. 14869)+brown        sugar juice 19° Brix+Vortexx ESF or Whisper V.    -   Four hours after biocide addition in the flask (T=4)→10% of        yeast+Lactobacillus infection (ATCC Deposit No. 14869)+brown        sugar juice 19° Brix+Vortexx ESF or Whisper V.

Fermentation samples were plated in two types of agar medium before andafter biocide addition aiming to count yeast and bacteria reductionafter treatment. The objective of biocide treatment is to reducebacterial contamination at least 100 times or “x” (10²). Treatments weredone in duplicate.

Fermentations where were added 150 and 200 ul of Vortexx ESF nobacterial growing were observed in a dilution of 10-3 after 1 and 4hours of biocide addition. For this reason, the number of bacteria inthese samples was lower than 1×10³ cells/mL. The results using VortexxESF (peracetic acid+octanoic acid) are shown in FIG. 2 and Table 1 asdescribed herein.

-   -   T0 Control—no biocide addition    -   T1 Control—no biocide addition. After 1 hour of fermentation.    -   T4 Control—no biocide addition. After 4 hours of fermentation.    -   T1—50 ul of biocide in 100 ml of fermentation (500 ppm of        product)—after 1 hour of biocide addition.    -   T4—50 ul of biocide in 100 ml of fermentation (500 ppm of        product)—after 4 hours of biocide addition.    -   T1—100 ul of biocide in 100 ml of fermentation (1000 ppm of        product)—after 1 hour of biocide addition.    -   T4—100 ul of biocide in 100 ml of fermentation (1000 ppm of        product)—after 4 hours of biocide addition.    -   T1—150 ul of biocide in 100 ml of fermentation (1500 ppm of        product)—after 1 hour of biocide addition.    -   T4—150 ul of biocide in 100 ml of fermentation (1500 ppm of        product)—after 4 hours of biocide addition.    -   T1—200 ul of biocide in 100 ml of fermentation (2000 ppm of        product)—after 1 hour of biocide addition.    -   T4—200 ul of biocide in 100 ml of fermentation (2000 ppm of        product)—after 4 hours of biocide addition.

TABLE 1 VORTEXX ESF - BACTERIA B0 V T0 control 5.00E+06 B1 V T1  50 ul3.35E+06 B1 V T4  50 ul 3.90E+06 B0 V T0 control 4.50E+06 B2 V T1  50 ul2.65E+06 B2 V T4  50 ul 3.30E+06 B3 V T1 100 ul 1.85E+05 B3 V T4 100 ul7.00E+04 B4 V T1 100 ul 2.25E+06 B4 V T4 100 ul 7.50E+04 B5 V T1 150 ul1.00E+03 B5 V T4 150 ul 1.00E+03 B6 V T1 150 ul 5.00E+03 B6 V T4 150 ul1.00E+03 B7 V T1 200 ul 1.00E+03 B7 V T4 200 ul 1.00E+03 B8 V T1 200 ul1.00E+03 B8 V T4 200 ul 1.00E+03 B9 V T1 control 5.90E+06 B9 V T4control 4.50E+06 B10 V T1 control 5.95E+06 B10 V T4 control 4.60E+06

Vortexx ESF demonstrates efficacy as a biocide to use during ethanolfermentation. It was observed a bacterial reduction in at least 10 times1 hour after add 100 ul of Vortexx ESF in 100 ml of fermentation and atleast 100 times 4 hours after biocide addition. We also observed thatbacterial reduction after add 150 ul and 200 ul of Vortexx ESF in 100 mlof fermentation was 1000× lower comparing to control samples.

In addition, no significantly yeast reduction was observed after anydosage of Vortexx ESF, as shown in FIG. 3 in Table 2. Accordingly themethods of the invention show a clear benefit in the ethanolfermentation industry without causing any detrimental reduction in yeastcell numbers that would impair the efficiency of fermentation. However,according to preferred aspects of the invention, the biocidefermentation would preferably not be conducted using a peracidcomposition including peracetic acid, e.g. Vortexx ESF (peraceticacid+octanoic acid). This is due in part to the peracetic acidintroducing an acetic acid which can result in false positive tests onquality control applications; the organic acid appears on plant qualitycontrol tests (e.g. Gas Chromatography) as a false indicator ofinfection with acid-forming bacteria.

TABLE 2 VORTEXX ESF - YEAST L0 V T0 control 6.25E+08 L1 V T1  50 ul6.00E+08 L1 V T4  50 ul 6.00E+08 L0 V T0 control 6.50E+08 L2 V T1  50 ul8.50E+08 L2 V T4  50 ul 1.10E+09 L3 V T1 100 ul 5.50E+08 L3 V T4 100 ul1.00E+09 L4 V T1 100 ul 6.00E+08 L4 V T4 100 ul 8.50E+08 L5 V T1 150 ul7.00E+08 L5 V T4 150 ul 4.50E+08 L6 V T1 150 ul 6.00E+08 L6 V T4 150 ul4.00E+08 L7 V T1 200 ul 5.90E+07 L7 V T4 200 ul 6.30E+07 L8 V T1 200 ul1.20E+08 L8 V T4 200 ul 1.50E+08 L9 V T1 control 9.00E+08 L9 V T4control 7.50E+08 L10 V T1 control 7.50E+08 L10 V T4 control 8.00E+08

The results using Whisper V (quaternary ammonium compound) are shown inFIG. 4 and Table 3 as described herein. Fermentations where were added400 and 750 ul of Whisper V no bacterial growing were observed in adilution of 10-4. For this reason, the number of bacterial in thesesamples was lower than 1×10⁴ cells/mL.

-   -   T0 Control—no biocide addition    -   T1 Control—no biocide addition. After 1 hour of fermentation.    -   T4 Control—no biocide addition. After 4 hours of fermentation.    -   T1—100 ul of biocide in 100 ml of fermentation (1000 ppm of        product)—after 1 hour of biocide addition.    -   T4—100 ul of biocide in 100 ml of fermentation (1000 ppm of        product)—after 4 hours of biocide addition.    -   T1—200 ul of biocide in 100 ml of fermentation (2000 ppm of        product)—after 1 hour of biocide addition.    -   T4—200 ul of biocide in 100 ml of fermentation (2000 ppm of        product)—after 4 hours of biocide addition.    -   T1—400 ul of biocide in 100 ml of fermentation (4000 ppm of        product)—after 1 hour of biocide addition.    -   T4—400 ul of biocide in 100 ml of fermentation (4000 ppm of        product)—after 4 hours of biocide addition.    -   T1—750 ul of biocide in 100 ml of fermentation (7500 ppm of        product)—after 1 hour of biocide addition.    -   T4—750 ul of biocide in 100 ml of fermentation (7500 ppm of        product)—after 4 hours of biocide addition.

In an aspect of the invention a quaternary ammonium compound, e.g.Whisper V, is not a preferred compositions for use according to theinvention. This is due to the preferred aspects of using compositionsthat lack any contamination residues in a subsequent animal feed productgenerated from the ethanol fermentation process.

TABLE 3 WHISPER V - BACTERIA B0 W T0 control 1.50E+05 B1 W T1 100 ul5.00E+04 B1 W T4 100 ul 1.50E+05 B0 W T0 control 1.00E+05 B2 W T1 100 ul1.00E+05 B2 W T4 100 ul 1.00E+05 B3 W T1 200 ul 5.00E+04 B3 W T4 200 ul1.50E+05 B4 W T1 200 ul 2.00E+05 B4 W T4 200 ul 2.50E+05 B5 W T1 400 ul1.00E+05 B5 W T4 400 ul 10000 B6 W T1 400 ul 1.00E+04 B6 W T4 400 ul10000 B7 W T1 750 ul 1.00E+04 B7 W T4 750 ul 10000 B8 W T1 750 ul1.00E+04 B8 W T4 750 ul 10000 B9 W T1 control 4.00E+05 B9 W T4 control1.00E+05 B10 W T1 control 4.00E+05 B10 W T4 control 2.00E+05

It was not possible to affirm what is the best dosages of Whisper V intofermentation flasks to control bacterial infection because was notpossible to count bacteria reduction after add 400 and 750 ul of thisbiocide into 100 ml of fermentation test. The minimum dilution plated(10-4), there was no bacterial growth (estimated bacterial cell numberwas lower than 10⁴ cells/ml). No significantly yeast reduction wasobserved after any dosage of Whisper V as shown in FIG. 5 and Table 4.

TABLE 4 WHISPER V - YEAST L0 W T0 control 1.80E+08 L1 W T1 100 ul6.50E+08 L1 W T4 100 ul 3.50E+08 L0 W T0 control 2.70E+08 L2 W T1 100 ul6.00E+08 L2 W T4 100 ul 6.50E+08 L3 W T1 200 ul 4.50E+08 L3 W T4 200 ul4.50E+08 L4 W T1 200 ul 8.00E+08 L4 W T4 200 ul 5.50E+08 L5 W T1 400 ul6.00E+08 L5 W T4 400 ul 6.00E+08 L6 W T1 400 ul 4.00E+08 L6 W T4 400 ul3.50E+08 L7 W T1 750 ul 3.50E+08 L7 W T4 750 ul 1.50E+08 L8 W T1 750 ul2.50E+08 L8 W T4 750 ul 1.00E+08 L9 W T1 control 5.50E+08 L9 W T4control 9.50E+08 L10 W T1 control 1.10E+09 L10 W T4 control 7.50E+08

Example 2

Additional testing was completed to analyze the efficacy of Vortexx ESF(peracetic acid+octanoic acid) (Ecolab, Inc.) and Octave (peroxyoctanoicacid) (Ecolab, Inc.) mixed peracid compositions for controllingbacterial contaminants in ethanol fermentation processes. As set forthin Example 1 there are inherent disadvantages to employing a peracidcomposition including peracetic acid/acetic acid and therefore there aresuperior peracid and mixed peracid compositions for use according to themethods of the invention that do not employ the peracetic acid.

The methods of the testing included:

Reagents

Solutions of Vortexx and Octave were provided by the Ecolab, Inc. Theantibiotic Virginiamycin was purchased from SantaCruz Biotechnology.Ethanol Red yeast (Fermentis), and Lactobacillus fermentum were revivedfrom 10% glycerol stocks maintained at 70 C. Yeast was grown at 30 Cunder aerobic conditions in either liquid YPD medium or on YPD agarprior to use in propagations and fermentations. Bacteria were grown at30 C under anaerobic conditions in either liquid MRS broth, and or onMRS agar plates. Liquefied corn mash and gluco-amylase enzyme wasprovided by Illinois River Energy (Rochelle, Ill.).

Fermentations

Fermentations were conducted in Wheaton glass bottles placed in a 30 Crecirculating water bath atop an 8-position magnetic stir-plate. Eachbottle was sealed with a rubber septum cap and connected through ⅛″tubing to a respirometer instrument (Challenger Instruments), whichquantified the off-gassing of CO₂ during fermentation.

Corn mash fermentations were conducted in 0.5 L Wheaton bottles, eachcontaining the following typical components: liquefied corn mash (400grams), gluco-amylase (0.33 mL), 50% urea solution (0.67 grams), andpropagated yeast (20 mL). When antibiotics or mixed peracid productswere used they were added with 800 microliters of water at the bottom ofthe bottle before filling with corn mash. The water was intended tosimulate the CIP rinse water volume. Virginiamycin was used at 0.5 ppmor 1 ppm, and Vortexx or Octave was used between 2.1 and 30 percent inthe rinse water volume.

MRS broth fermentations were conducted in 0.125 L Wheaton bottles, eachcontaining the 100 mL of MRS broth and 4 mL propagated bacteria. Whenantibiotics or mixed peracid products were used they were added with 200microliters of water at the bottom of the bottle before filling with MRSbroth.

Measurement of Residual Sugars, Acids, and Alcohols

In preparation for HPLC analysis, completed fermentation samples werecentrifuged for 10 minutes at 1000 rpm and the supernatant filteredthrough 0.45 micron nylon syringe filters. Filtered samples were diluted1:50 in 2 mM H₂SO₄ mobile phase prior to injection into the column(Aminex HPX-87H (300×7.8 mm) with guard column). The column flow ratewas 0.4 mL/min, the temperature was 25 degrees Celsius, and the run timewas 50 minutes per sample. Analytes were detected using a refractiveindex detector (recorder range—512 uRIU/FS; integrator range—125 uRIU/V;temperature—30° C.; response time—0.5 seconds; polarity—positive;baseline shift—40 mV; sequence—standard).

Calibration of the HPLC instrument was performed using a fuel ethanolresidual saccharide mix purchased from Sigma Aldrich (#48468-U). A stocksolution was made by diluting the standard ten-fold in mobile phase.Refractive Index detectors tend to be fairly sensitive, thus it isrecommended diluting the standards and samples in the same mobile phaserunning through the column on the HPLC system. All subsequent standardsare created by making further dilutions of the stock standard solutionafter it has stirred for a sufficient amount of time (at least 15minutes). The eight compounds monitored by HPLC along with theirrespective retention times are shown in Table 5. Standard K is the stockstandard dilution, and all letters from B to J are further dilutions ofthis standard. The dilution factors were multiplied by the actualconcentration listed on the COA, which allowed the concentration columnsfor each compound to be determined. Since resolution between dextrin,maltotriose and maltose is relatively poor, the height of thesecompounds provided a more accurate determination of theirconcentrations. For all other compounds, area is used to develop acalibration curve.

TABLE Standard dilutions Standards (6400-pg107) Dextrin - 9.9 min B0.000220162 Maltotriose - 10.7 min C 0.000717274 Maltose - 11.5 min D0.002148433 D-Glucose - 13.6 min E 0.004302416 L-(+)-Lactic Acid - 19.1min F 0.007912708 Glycerol - 20.1 min G 0.012416336 Acetic Acid - 23.5min H 0.017314458 Ethanol - 31.8 min I 0.024019447 J 0.048444897 K0.114749254 Conc (ppm) Height Rf Dextrin - 9.9 min y = 0.3976x − 4.18097.2 1.191 6.007788679 23.3 5.830 3.998522966 69.8 23.819 2.931444198139.8 50.639 2.761281395 257.2 95.872 2.682357805 403.5 156.4052.580038531 562.7 219.514 2.563480689 780.6 305.321 2.556758388 1574.5621.484 2.533386457 3729.4 1478.932 2.52165127 Average - 3.179451012Maltotriose - 10.7 min y = 0.5607x − 0.0825 2.2 1.29 1.69619241 7.1 4.011.770824861 21.3 12.00 1.772161785 42.6 23.90 1.782023304 78.3 43.851.786286506 122.9 69.08 1.779411238 171.4 95.81 1.789113118 237.8 133.131.786127597 479.6 268.01 1.789535566 1136.0 637 1.782763044 Average -1.772408487 Maltose - 11.5 min y = 0.5128x + 0.0199 4.4 2.30 1.89613505614.2 7.28 1.950826289 42.5 21.97 1.936406204 85.2 43.83 1.943818415156.7 80.36 1.949718961 245.8 126.49 1.943580178 342.8 175.511.953281395 475.6 243.96 1.94943864 959.2 491.06 1.953339725 2272.0 11651.949694232 Average - 1.941838318 Conc (ppm) Area Rf D-Glucose - 13.6min y = 15703x + 13136 4.4 87063 5.03225E−05 14.3 234463 6.08784E−0542.8 687080 6.22254E−05 85.6 1360558 6.29287E−05 157.5 24829336.34181E−05 247.1 3915826 6.30991E−05 344.6 5425713 6.35046E−05 478.07520488  6.3558E−05 964.1 15101833 6.38369E−05 2283.5 358881206.36286E−05 Average - 6.15302E−05 L-(+)-Lactic Acid - 19.1 min y =10233x − 2285.2 0.7 6583 0.000100332 2.2 22005 9.77878E−05 6.4 623330.000103401 12.9 128172 0.000100703 23.7 240902 9.85385E−05 37.2 3795069.81513E−05 51.9 534629 9.71578E−05 72.1 732351 9.83932E−05 145.31476872 9.84071E−05 344.2 3523625  9.7697E−05 Average - 9.92079E−05Glycerol - 20.1 min y = 12718x + 25535 2.2 34346 6.34603E−05 7.1 1045366.79288E−05 78.3 1105441 7.08639E−05 122.9 1593811 7.71244E−05 171.42185621 7.84277E−05 237.8 3026638 7.85666E−05 479.6 6095898 7.86766E−051136.0 14487479 7.84138E−05 Average - 6.80889E−05 Acetic Acid - 23.5 miny = 6606.4x + 1837.9 0.7 5127 0.000133119 2.2 15150 0.000146769 6.744331 0.000150237 13.3 92391 0.000144359 24.5 166629 0.00014721  38.5257424 0.000149522 53.7 356104 0.000150728 74.5 495375 0.000150311 150.2988787 0.000151882 355.7 2353399 0.000151153 Average - 0.000147126Ethanol - 31.8 min y = 6158.6x − 42237 26.2 155449 0.000168398 85.3490636 0.000173823 255.4 1512846 0.000168853 511.6 3048365 0.000167814940.8 5741237 0.000163871 1476.3 9071669 0.000162738 2058.7 126493960.00016275  2855.9 17570209 0.000162543 5760.1 35417028 0.000162636Average - 0.000165936

The objectives of the analysis included measuring residual levels ofperacid and hydrogen peroxide after exposure to fermentable substrateand to yeast; inoculating contaminating bacterial species with thefermentable substrate and determining the reduction in viable cellsafter exposure to the mixed peracid products; and investigation of anynegative impact of peracids on normal fermentation performance and yeastactivity.

Results

The concentrations of peracid and hydrogen peroxide actives at variousstages of filling the fermentation tank were analyzed. This analysisused the initial active concentrations reported in the catalog sheetsfor Vortex and Octave. It was also assumed that 5.0% Vortexx or 2.1%Octave were added to the CIP rinse water. The rinse water volume wasassumed to be 0.2% of the fermenter tank volume. The methods simulatedthe dilution of the sanitizing solution of the peracid and hydrogenperoxide actives (peracid composition) in the tank due to filling withmash. As a result, the concentrations were constantly diluted in themash as the tank fills. This is shown below in Table 6, illustrating thecombined effect of dilution and reaction of the actives with sugars inthe mash. The table demonstrates the dynamic occurring with mashaddition.

Prior research (Urea Hydrogen Peroxide Reduces the Number ofLactobacilli, Nourishes Yeast, and Leaves No Residue in the EthanolFermentation. N. V. Narendarath, K. C. Thomas, M. W. Ingledew (2000).Appl. Env. Microbiology, Vol. 66, pp. 4187-4192) found that 600 ppm ofhydrogen peroxide and 2 hours of contact time in the absence of yeastwas required for effective bacterial control.

As can be seen in Table 6 (dilution corrected active concentrations),the peracid and hydrogen peroxide concentrations are well below 100 ppmeven when the fermenter is only 10% full, demonstrating that thedilution of the CIP rinse volume with fermentable substrate is achallenge associated with the invention. However, the actualconcentrations are likely to be much less than those reported in Table6. Further reductions in the active concentrations will certainly occuras the peracids attack the highly loaded organic matter in thefermentable substrate, and as the yeast degrade the hydrogen peroxide.

TABLE 6 Vortexx Octave tank level tank volume Conc. [PAA] ppm [H202] ppmConc. [POA] ppm [H202] ppm 0.00% 1000  5.00% 2200.0 3450.0  2.10% 197.41596.0 10.00% 51000  0.10% 43.1 67.6  0.04% 3.9 31.3 20.00% 1010000.050% 21.8 34.2 0.021% 2.0 15.8 30.00% 151000 0.033% 14.6 22.8 0.014%1.3 10.6 40.00% 201000 0.025% 10.9 17.2 0.010% 1.0 7.9 50.00% 2510000.020% 8.8 13.7 0.008% 0.8 6.4

Survival of Vortexx in Corn Mash

Corn mash from a local dry-grind ethanol plant was diluted 1:10 withwater. Vortexx was mixed into the 10-fold diluted corn mash at 1:1000ratio. PAA test strips from LaMotte were used to track the survival ofVortexx in the corn mash. After 60 minutes, the Vortexx was almostcompletely consumed by the corn mash.

As a control, pure water was treated with the same Vortexx solution at1:1000 ratio. No detectable consumption of Vortexx was observed over thesame 60 minute period.

This experiment demonstrates that corn mash, even at 10% of its normalsolids concentration, possesses an apparent demand for the oxidizingchemistries in Vortexx. Thus, the decay rate shown here could beexpected to increase by 10-fold when using full strength corn mash.Numerous additional factors affect decay rate, including for example,diffusion rate of the sanitizing solution in the viscous mash beingadded. Peracid Chemistries for Controlling Lactobacilli in MRS Broth

MRS broth, which contains only 0.6% solids compared to typical liquefiedcorn mash at 30% solids, represents a relatively simple test of theeffectiveness of the peracid chemistries for controlling Lactobacillusgrowth. A pure culture of L. fermentum was inoculated into MRS broth ata starting concentration of 1×10⁵ cell/mL. The MRS broth with bacteriawas split into four different bottles. The first bottle contained noantibiotic or peracid. The second and third bottles contained 5% Vortexxor 5% Octave, respectively, in the rinse water. The fourth bottlecontained 1 ppm virginiamycin. All bottles were placed monitored viarespirometry under identical conditions. As seen in FIG. 6, bottles 1,2, and 3 produced identical amounts of CO₂ with exactly the samekinetics. Bottle 4 failed to produce any CO₂ and exhibited no bacterialgrowth. These observations indicate that applying peracid chemistries inthe rinse water will not control bacterial growth alone, and thereforewould need to be combined with out cleaning steps (e.g. CIP/COP, etc.).

Impact of Peracid Chemistries on Yeast in Corn Mash

Several corn mash fermentation experiments were performed to assess thepotential impact of the peracid chemistries on yeast activity. Threesets of fermentation experiments were performed, and in each set eightdifferent fermentations were conducted to allow for variation of thebacterial innocula and anti-bacterial control strategies.

In the first experiment, 5% Vortexx or 5% Octave in the rinse water hadabsolutely no impact on the total volume of CO₂ produced or the rate ofCO₂ production (FIG. 7).

HPLC analysis of the completed fermentation samples also revealed nodifferences among the eight samples in residual sugars, acids, andalcohol at the end of fermentation (Table 7).

TABLE 7 Dextrin Maltitriose Maltose Glucose Lact Acd Glycerol AceticAcid Ethanol Sample Conc (%) Conc (%) Conc (%) Conc (%) Conc (%) Conc(%) Conc (%) Conc (%) 1 1.03 ND < 0.20 0.37 0.44 0.6 1.98 0.1 14.55 21.03 ND < 0.20 0.36 0.43 0.56 1.99 0.1 14.71 3 1.05 ND < 0.20 0.38 0.410.61 1.99 0.1 14.69 4 1.04 ND < 0.20 0.38 0.39 0.61 1.99 0.09 14.66 51.03 ND < 0.20 0.39 0.42 0.65 1.99 0.09 14.6 6 1.04 ND < 0.20 0.37 0.420.59 1.99 0.1 14.62 7 1.03 ND < 0.20 0.38 0.42 0.62 1.98 0.09 14.55 81.03 ND < 0.20 0.38 0.4 0.62 1.98 0.09 14.61

In the second experiment, 10% Vortexx or 30% Vortexx in the rinse waterwere shown to slow down the initial rate of CO₂ production, butultimately there was no impact on the total volume of CO₂ produced (FIG.8).

HPLC analysis of the completed fermentation samples also revealed nodifferences among the eight samples in residual sugars, acids, andalcohol at the end of fermentation (Table 8).

TABLE 8 Dextrin Maltitriose Maltose Glucose Lact Acd Glycerol AceticAcid Ethanol Sample Conc (%) Conc (%) Conc (%) Conc (%) Conc (%) Conc(%) Conc (%) Conc (%) 1 0.92 ND < 0.20 0.36 0.18 0.63 1.78 0.08 13.84 20.92 ND < 0.20 0.37 0.19 0.6 1.72 0.11 13.96 3 0.93 ND < 0.20 0.36 0.160.62 1.84 0.1 13.93 4 0.93 ND < 0.20 0.37 0.17 0.67 1.79 0.08 14.02 50.93 ND < 0.20 0.38 0.19 0.63 1.72 0.09 14.02 6 0.94 ND < 0.20 0.39 0.20.64 1.82 0.09 13.93 7 0.92 ND < 0.20 0.41 0.2 0.71 1.83 0.11 13.88 80.93 ND < 0.20 0.41 0.19 0.67 1.74 0.09 13.92

The inventions being thus described, it will be obvious that the samemay be varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the inventions and all suchmodifications are intended to be included within the scope of thefollowing claims. Since many embodiments can be made without departingfrom the spirit and scope of the invention, the invention resides in theclaims.

What is claimed is:
 1. A method for reducing and/or eliminatingmicrobial populations of yield loss organisms in a biofuel fermentationsystem comprising: applying a peracid composition to sanitize afermentation system, wherein the peracid composition comprises a shortchain peracid and a sequestrant wherein the fermentation systemcomprises one or more fermentation vessels, pipes and/or components;providing or obtaining a mash source in said fermentation system; andreducing and/or eliminating a microbial population of yield lossorganisms in said fermentation system.
 2. The method of claim 1, whereinthe yield loss organisms are lactic acid bacteria and/or acetic acidbacteria.
 3. The method of claim 1, wherein the peracid compositioncomprises peroxyformic acid, formic acid, hydrogen peroxide, asequestrant, and water and does not include acetic acid and/or peraceticacid.
 4. The method of claim 1, wherein the peracid compositioncomprises from about 0.5 wt-% to about 5 wt-% peroxyformic acid, fromabout 1 wt-% to about 10 wt-% formic acid, from about 5 wt-% to about 97wt-% water, from about 0 wt-% to about 20 wt-% anionic surfactant, fromabout 0 wt-% to about 10 wt-% oxidizing agent; about 0 wt-% to about 35wt-% inorganic acid, and from about 0 wt-% to about 5 wt-% sequestrant.5. The method of claim 1, further comprising fermenting a fermentablemash in the presence of at least a portion of said peracid composition,and yeast in a vessel to produce ethanol and a solids content, whereinsaid peracid composition controls growth of bacteria in the mash withoutreducing yeast population; and distilling the fermented mash to separateat least a portion of the ethanol from said solids content.
 6. A methodfor reducing yield loss in ethanol fermentation processes comprising:applying a peracid composition to sanitize a fermentation system,wherein the peracid composition comprises a short chain peracid and asequestrant; fermenting a mash in the presence of at least a residualportion of said peracid composition and yeast in a vessel of saidfermentation system to produce ethanol and a solids content; reducing amicrobial population of lactic acid bacteria and/or acetic acidbacteria; and distilling the fermented mash to separate at least aportion of the ethanol from said solids content, wherein theantimicrobial efficacy of said peracid composition does not interferewith yeast fermentation.
 7. The method of claim 6, wherein said peracidcomposition is applied to said fermentation system for providingsanitizing of said system without requiring a rinse step of saidfermentation system, and wherein said portion of said peracidcomposition is a residual amount remaining in said vessel aftersanitizing.
 8. The method of claim 6, wherein the peracid compositioncomprises peroxyformic acid, formic acid, hydrogen peroxide, asequestrant, and water.
 9. The method of claim 6, wherein the lacticacid bacteria and/or acetic acid bacteria are Lactobacillus species,Acetobacter species or combinations thereof.
 10. A method for replacingantibiotics in biofuel fermentation processes comprising: replacing anantibiotic used in a fermentation process to reduce and/or eliminateLactobacillus and/or Acetobacter species with a peracid composition forapplication to a fermentation system; and introducing the peracidcomposition to a mash source, wherein the peracid composition comprisesfrom about 0.0005 wt-% to about 5 wt-% peroxyformic acid, from about 1wt-% to about 10 wt-% formic acid, from about 5 wt-% to about 97 wt-%water, from about 0 wt-% to about 20 wt-% anionic surfactant, from about0 wt-% to about 10 wt-% oxidizing agent; about 0 wt-% to about 35 wt-%inorganic acid, and from about 0 wt-% to about 5 wt-% sequestrant, andwherein the peracid composition does not interfere with yeastfermentation in a fermentation process; wherein the peracid compositiondoes not introduce to the fermentation system a source of acetic acidand/or peracetic acid.
 11. The method of claim 10, wherein the peracidcomposition comprises from about 0.5 wt-% to about 5 wt-% peroxyformicacid, from about 1 wt-% to about 10 wt-% formic acid, from about 5 wt-%to about 97 wt-% water, from about 1 wt-% to about 20 wt-% anionicsurfactant, from about 5 wt-% to about 10 wt-% oxidizing agent; about 15wt-% to about 35 wt-% inorganic acid, and from about 1 wt-% to about 5wt-% sequestrant.
 12. The method of claim 10, further comprisingfermenting the mash source in the presence of at least a portion of saidperacid composition, and yeast in a vessel to produce ethanol and asolids content, wherein said peracid composition controls growth ofbacteria in the mash without reducing yeast population; and distillingthe fermented mash to separate at least a portion of the ethanol fromsaid solids content.
 13. The method of claim 12, further comprisingobtaining a distillers dried grains product from said solid contentscontaining less than about 1000 ppm of the peracid composition.
 14. Themethod of claim 10, wherein the peracid composition is introduced to thefermentation process within a clean-in-place application to clean thefermentation systems employed for the fermentation process.
 15. Themethod of claim 14, wherein the peracid composition is introduced eitherupstream from a fermentation vessel or directly into the fermentationvessel.
 16. The method of claim 14, wherein the peracid composition or aportion of the peracid composition is retained in the fermentationvessel after the clean-in-place application and used for reducing and/oreliminating the Lactobacillus and/or Acetobacter species during thefermentation process.
 17. The method of claim 10, wherein the peracidcomposition is introduced to the fermentation process as a directadditive to the mash source.
 18. The method of claim 10, wherein theperacid compositions have low or no adverse environmental impact. 19.The method of claim 1, wherein the peracid composition comprisesperoxyformic acid and a medium chain peracid.